Note: Descriptions are shown in the official language in which they were submitted.
CA 02891445 2015-05-14
TITLE OF THE INVENTION
Method And Composition For Producing Enhanced Anti-Inflammatory/Anti-catabolic
And
Regenerative Agents From Autologous Physiological Fluid
FIELD OF THE INVENTION
The present invention is directed to a method and composition for the
treatment of damaged
and/or injured connective tissues including chronic tendinosis, chronic muscle
tears, chronic
degenerative joint conditions such as osteoarthritis as well as chronic
inflammatory skin diseases
including, atopic dermatitis and chronic wounds.
BACKGROUND OF THE INVENTION
Osteoarthritis is degenerative joint disease characterized by cartilage damage
and synovial
inflammation. Previous data refer to changes to a molecular inflammatory
cascade that lead to a
destruction of cartilage macromolecules and irreversible morphological
changes'. Considerable
evidence has shown that IL-1, Tumor Necrosis Factor-alpha, IL-6,8 are
predominant catabolic
pro-inflammatory molecules playing a major role in pathogenesis of
osteoarthritisl. These
cytokines are produced by activated synoviocytes, mononuclear cells or by
articular cartilage
itself and their catabolic effect could be successfully blocked by inhibitory
cytokines such as IL-
4,10,13 and IL-lral.
Similar inflammatory and catabolic pathways are involved in the pathogenesis
of chronic
tendonitis2 and chronic muscle tear healing failure3. Tendon cells subjected
to continuous
damage by producing increased levels of IL-1,6, metalloproteinases (MMPs) and
other catabolic
molecules2. Pro-inflammatory cytokines IL-1 and TNF-alpha are involved in
pathogenesis of
chronic myositis3 as well. Atopic dermatitis (eczema) is considered as the
most common
relapsing inflammatory skin conditions. Chronic wound (including diabetic
wound) is a wound
that does not heal within three months clue to poor circulation, neuropathy,
immune disorders and
complications of systemic illnesses, age, and repeated trauma. All mentioned
conditions are
CA 02891445 2015-05-14
characterized by disturbing cell signaling via cytokines and lost
extracellular matrix (ECM) that
forms the largest component of the dermal skin layer. Targeting special
inflammatory and
catabolic molecular pathways can have a beneficial therapeutic effect for
inflammatory
pathologies. This effect could be achieved by using therapeutically active
proteins. Presently, the
pharmaceutical industry employs high cost molecular genetic technologies for
recombinant
protein production such as insulin, interferons, blood clotting factors, etc.
However, these
methods of recombinant protein generation include the expression of human
genes in a bacterial
cell. The patterns of post-translation protein modification including
glycosylation may be
different than those natural occurring in humans. This may result instability
of the product in the
human environment, decreasing of biological function or immune response
provocation.
Additionally, the cost of the final recombinant product is extremely high.
There is therefore a need for an alternative product for treating degenerative
joint disease in
humans that is safe effective, stable, regenerative and cost effective.
SUMMARY OF THE INVENTION
The invention is directed to a bioactive composition for treating damaged
and/or injured
connective tissues, chronic tendinosis, chronic muscle tears and/or chronic
degenerative joint
conditions such as osteoarthritis, and skin inflammatory disorders. The
invention is also directed
to a method for making the composition. The composition includes a
regenerative component
and an anti-inflammatory component/anti-catabolic component. The anti-
inflammatory
component of the present invention is also an anti-catabolic component. For
the purposes of the
present invention the terms anti-inflammatory component and anti-catabolic
component can be
used interchangeably. Preferably, the regenerative component includes
autologous platelet rich
plasma (PRP). Whereas most PRP preparation protocols include an activation
step resulting in
the immediate release of growth factors and cytokines from platelets, the
present invention
provides a use of a non-activated PRP component for future slow activation of
injected
composition by surrounding tissue.
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The anti-inflammatory/ anti-catabolic component comprises preferably IL-lra,
an IL-1 receptor
antagonist containing autologous serum. In addition, the anti-inflammatory/
anti-catabolic
component preferably comprises an increased level of tissue inhibitors of
metalloproteinases
(TIMPs).
According to one aspect of the present invention, there is provided a method
of producing an
autologous composition for treating damaged and/or injured connective tissues,
chronic
tendinosis, chronic muscle tears and/or chronic degenerative joint conditions
and skin
inflammatory disorders in a mammal comprising the following steps:
A) preparing an anti- inflammatory/ anti-catabolic component of the autologous
composition
comprising TIMPs and IL-lra, said step of preparing the anti-inflammatory/
anti-catabolic
component comprising the following steps: i) collecting blood from the mammal;
ii) delivering
the blood to a tube; iii) incubating the blood at a temperature of from about
37 C to about 39 C
for about 24 hours; iv) centrifuging the blood to separate the blood into a
supernatant component
and a cellular fraction; and v) collecting the supernatant component;
B) preparing a regenerative component of the autologous composition comprising
the following
steps: i) collecting blood from the mammal; ii) delivering the blood to a tube
in the presence of
about 4% citric acid; iv) centrifuging the whole blood to separate the
platelet rich plasma
component; and v) collecting the platelet rich plasma component; and
C) mixing the supernatant component of the anti- inflammatory/ anti-catabolic
component with
the platelet rich plasma component to provide the autologous composition.
According to another aspect of the present invention, there is provided an
autologous
composition for treating damaged and/or injured connective tissues, chronic
tendinosis, chronic
muscle tears and/or chronic degenerative joint conditions and skin
inflammatory disorders in a
mammal produced by the method of the present invention.
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According to another aspect of the present invention, there is provided an
autologous
composition for treating damaged and/or injured connective tissues, chronic
tendinosis, chronic
muscle tears and/or chronic degenerative joint conditions and skin
inflammatory disorders in a
mammal, the composition comprising an anti-inflammatory/anti-catabolic, said
anti-
inflammatory/anti-catabolic component comprising TIMPs and IL-lra. The
composition further
comprises a regenerative component comprising platelet rich plasma.
According to yet another aspect of the present invention, there is provided a
use of the
autologous composition of the present invention for the treatment of damaged
and/or injured
connective tissues, chronic tendinosis, chronic muscle tears and/or chronic
degenerative joint
conditions and skin inflammatory disorders in a mammal.
According to yet another aspect of the present invention, there is provided a
method of treating a
patient for damaged and/or injured colnective tissues, chronic tendinosis,
chronic muscle tears
and/or chronic degenerative joint conditions and skin inflammatory disorders
comprising the
following steps:
collecting blood from the patient;
delivering the blood to a tube;
incubating the blood at a temperature of from about 37 C to about 39 C for
about 24 hours;
centrifuging the blood to separate the blood into a supernatant component and
a cellular fraction;
collecting the supernatant component;
preparing a regenerative component of the autologous composition comprising
the following
steps:
collecting blood from the mammal;
delivering the blood to a tube in the presence of about 4% citric acid;
centrifuging the blood to separate a platelet rich plasma component from a
whole blood;
collecting the platelet rich plasma component;
mixing the supernatant component with the platelet rich plasma component to
provide the
autologous composition; and
administering the autologous composition to the patient.
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DESCRIPTION OF THE DRAWINGS
Figure 1 a plot of IL-1ra concentration in pg/ml versus time showing a
comparison of the level of
IL-1ra antagonist protein in the human serum samples with varying incubation
conditions
including stationary and rocking at different time points.
Figure 2 a plot of IL-Ira concentration in pg/ml versus time showing a
comparison of the level of
IL-1ra antagonist protein in the human serum samples at different time points
in the presence of
air, Ca ++ (in Phosphate Buffered Saline, PBS) and different concentration of
serum.
Figure 3 is a Doppler ultrasound image of a right paraspinal area of a patient
before treatment (a)
and normal post-treatment condition (b).
Figure 4 is a Doppler ultrasound image showing Chronic Achilles tendinosis is
characterized by
excessive hyperemia (a, pre-treatment status), that was resolved as a result
of autologous
composition treatment (b, post-treatment imaging).
Figure 5 is a graph showing an average of concentration level of TIMP 1, TIMP
2 and TIMP 4
before and after 24h incubation;
Figure 6 is a graph showing an average baseline of joint pain according to the
VAS scale in the
eight patients prior to and after injections;
Figure 7 is a plot showing point values according to the WOMAC index for
average levels of
pain, stiffness and daily activity capabilities among the eight patients
tested;
Figure 8 shows results in the form of a graph providing a breakdown of the
average results after
the first, second and third injections and two and three months follow ups;
and
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Figure 9 is graph showing a comparison between the average concentration level
of PGDF in the
human serum samples before and after 24h incubation.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a composition comprising an autologous
platelet-rich plasma
component and an autologous anti-inflammatory/ anti-catabolic component serum
enriched by
bioactive proteins having a synergistic anti-inflammatory/ anti-catabolic,
proliferative, tissue
remodeling and regenerative effects.
The composition includes the following therapeutically active proteins: IL-
lra6, IL-47, IL-108'9,
IL-1310, PDGF11, TGF-131 '11 and VEGF12'13,14.
IL-lra is secreted by monocytes, adipocytes and epithelial cells.
Therapeutically effective
concentrations of this protein are ac' i,.wed by incubating human monocytes at
about 37 C for
about 24h17. IL-4,10,13, PDGF, TGF- f3 are the content of platelets a-granules
and are delivered
in the PRP component. Employing the synergistic effect of mentioned proteins
leads to
generation of a potent bio-active autologous product. Thus, a combination of
fresh-prepared
PRP as a source of regenerative biological factors and anti-inflammatory
cytokines and growth
factors, and the anti-inflammatory component comprising cultured autologous
serum as a source
of IL-1 inhibitor provides a powerful and cost-effective autologous
therapeutic agent for
treatment of degenerative conditions like osteoarthritis, chronic tendinosis
and chronic muscle
tears as well as skin inflammatory disorders.
It has been surprisingly discovered that the method of producing the anti-
inflammatory/ anti-
catabolic component of the present invention leads to the production of an
increased level of
tissue inhibitors of metalloproteinases (TIMPs). Matrix metalloproteinases
MMPs are believed
to cause joint destruction when in an active state. TIMPs neutralize active
MMPs thereby
providing an additional anti-catabolic benefit which is synergistic with IL-
lra.
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The method of the present invention for producing an autologous composition
for the treatment
of osteoarthritis, chronic tendinosis and chronic muscle tear as well as skin
inflammatory
disorders preferably comprises the step of collecting a mammal's blood by an
aseptic technique.
Preferably the mammal is a human. The site of venipuncture and the surface of
the collection
tubes is cleaned with a 2 percent tincture of iodine solution. Before any
cleansing of the site is
begun, the patient is asked about any allergy to iodine. The tube covers are
cleaned with 70%
alcohol solution also to avoid possible contamination before blood collection.
The composition of the present invention is preferably prepared by culturing
blood at a
temperature of preferably from about 37 C to about 39 C. Most preferably the
temperature is
between 37 C and 38 C. The blood is incubated for about 24 hours for IL- 1 ra
extracellular
enrichment and preferably for the production of TIMPs. In a particularly
preferred embodiment,
the incubation can be in sterile glass tubes (Coviden) or polystyrene (BD)
vacutainer tubes with
no additives. The invention provides in a further preferred embodiment the
incubation of an
autologous physiological fluid, preferably blood, on a rocker platform (24
rpm) or in static
conditions. Preferably incubation is carried out in static conditions as shown
in Figure 1.
The present invention preferably provides for the culturing of blood in the
presence of 0.64-0.72
mM Ca ++ to facilitate IL-1ra production15. It is possible and advantageous in
a particularly
preferred embodiment to dilute cultured blood with sterile CaC1 solution
containing 0.64-0,72
mM Ca ++ in 9:1 proportion by adding the solution using a sterile syringe and
needle directly to
the tube with blood before the incubation (lcc of the CaC1 solution to 9cc
whole blood) (Figure
2). An equal part of sterile air may be added to the sterile tubes containing
the blood to expose
the culture to atmospheric air for increasing IL-Ira production (Figure 2). In
a particularly
preferred embodiment, the air will be passed through 0.22um MillexGP filter
using a sterile
syringe and needle directly to the tube with the blood before the incubation.
The cultured blood is then subjected to centrifugation to separate the
supernatant component
from the cellular fraction. The centrifugation is carried out for about 10-20
minutes at about
4000-10000 rpm. Preferably the centrifugation is carried out for 10 minutes at
4000 rpm.
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The next step involves aspirating the supernatant and dividing it into
aliquots for future
processing using a sterile technique. The procedure is carried out in a
sterile environment
(laminar flow hood with HEPA filters). Three cc of the supernatant layer
containing biologically
active agents are carefully drawn by sterile syringe and needle. Prolonged
storage of IL-1ra
containing product is accomplished by freezing aliquots at about -20 C and
storing for up to 6
months or up to one year at about -70 C.
The preparation of the regenerative component comprising PRP then involves
drawing fresh
autologous physiological fluid into vacutainer tubes. This is preferably
carried out in the
presence of 4% citric acid. Preferably in a 9.5 parts of whole blood (9.5cc) :
0.5 (0.5cc) of 4%
citric acid ratio. The blood is then subjected to centrifugation preferably
for about 30 sec, at
about 7500 rpm to isolate the PRP fraction. The centrifugation parameters are
used in preferred
embodiments for the PRP preparation as a part of the final product for the
osteoarthritis and
chronic tendinosis treatment and skin disorders. The PRP fraction is drawn by
a sterile syringe
and needle under sterile conditions. In a particularly preferred embodiment
for the treatment of
chronic tear, a leukocyte buffy coat fraction is added to the PRP as an
additional VEGF source in
order to promote new blood vessel development in the affected site. The huffy
coat layer and
plasma is collected manually by sterile syringe and needle after whole blood
centrifugation as set
out above or using a commercially available Harvest SmartPrep system. The
regenerative
component comprising PRP is not subject to freezing or other storage. The
autologous
composition is to be administered to a patient promptly after mixing the
regenerative component
with the anti-inflammatory/ anti-catabolic component.
The anti-inflammatory/ anti-catabolic component comprising IL-1ra and
preferably TIMPs
containing blood is meted with the regenerative component comprising PRP
fraction preferably
in a 1:1 ratio to obtain the final product.
The product is injected for the future slow activation by tendon-derived
collagen16 and tissue-
derived thrombin.
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Examples
Example 1 - Comparison of extracellular IL-lra production upon different
culture conditions.
As shown graphically in Figure1, a comparison was carried out of the level of
IL-1ra antagonist
protein in the human serum samples that were exposed to incubation conditions
at different time
points including stationary versus rocking incubation. IL-1ra is secreted by
the activated blood
monocyte, macrophages. Such activation could be achieved by contact between
blood cells and
collection tubes' internal surfaces, through the use of an agitation process.
By increasing the
internal surface area exposed to the cellular component, the cell activation
process and bioactive
molecule secretion can be maximized. Peripheral blood from 10 healthy male and
female
volunteer donors (21 to 60 years old) was collected by venipuncture under
sterile conditions to
the sterile 10 ml glass tubes. One tube was manipulated according to a
standard procedure with
no incubation step (control sample). Samples were incubated for 3.5hrs, 7hrs,
and 24hrs at 37 C
with and without agitation on rocker platform (24 rpm). Incubated samples were
centrifuged for
minutes at 4,000 rpm and then filtered, and the final concentrations of IL-lra
s was compared
to those of unprocessed control sample:; (Oh) according to manufacture
protocol (http://www.bio-
rad .com/webroot/web/pdf/I sr/literatu re/10014905 .pdf). An One-way Anova
test reveals
significant increasing of IL-lra production in the serum after 24h of
incubation only; no
significant IL-lra concentration increasing was observed in 3.5h and 7h
incubated samples.
Additionally no significant difference was observed between stationary and
rocking incubation
conditions. IL-lra concentration was evaluated using MAGPIX Luminex
technology.
Figure 2 shows a comparison of the level of Il-lra antagonist protein in the
human serum
samples that were exposed to the following incubation conditions: 24h
incubation in the presence
of air, Ca++ (in Phosphate Buffered Saline, PBS) and different concentration
of serum. A One-
way Anova test reveals significant increasing of IL-lra production in the
serum after 24h of
incubation upon all mentioned conditions besides culturing in 50% diluted
blood.
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Example 2 - Case reports of patients diagnosed with osteoarthritis and chronic
tendinosis and
treated with the autologous composition of the present invention.
Case 1: S, 61 years.
Diagnosis: The patient reported bilateral insidious onset knee pain which
began a few
years prior and which had increased within the preceding 6 months. MRI of the
knees
shows severe OA of the knees: severe chondrosis of the medial compartment with
increased amount of full-thickness cartilage loss involving the right and a
femoral
condyle in the right knee and full thickness chondral loss involving the
posterior aspect of
the medial femoral trochlea with underlying edema in the right knee. One year
ago, the
patent had Cortisone injection IA, one month relief. Physical exam: Knees
Range of
Motion (ROM) is full, all ligaments are normal, small bilateral effusion
neurovascular
exam is normal. VAS is 60.
Treatment: Bilateral local autologous composition injections into knees x 3, a
week apart.
Result: After first injection reports significant improvement, VAS is 3. At
the time of the
injection -ROM full all ligaments are normal no joint line pain, no effusion.
Reports
strong pain reduction, VAS is " 0, comes back to physical activity. Three
months later, a
follow up exam shows that the patient is pain free.
Case 2: E, 64 years
Diagnosis: Active male presented with VAS 60 in the left hip. Pain with daily
activities
and significant impairments with walking for a long distance. MRI shows mild
OA in
both hips: bilateral hip joint degeneration and acetabular labral degenerative
tearing.
Physiotherapy treatment had limited success in terms of pain relief.
Treatment: US-guided local autologous composition injection into left hip x2,
a week
apart.
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Result: after 1st injection reports 85% improvement in pain (patient personal
assessment),
after second injection VAS is 1o; 5 months later VAS is 10.
Case 3: A, 70 years.
Diagnosis: A female patient presented with chronic pain (VAS is 6) tenderness
and
swelling in the left knee. She had much difficulty in walking, standing and
climbing
stairs. She has been to a physical therapist (6 visits), a chiropractor (6
visits) for help with
her knee pain with no results. MRI shows severe OA in left knee, there is the
narrowing
of the medial compartment due to full-thickness cartilage loss in the weight-
bearing
portion of the medial femoral condyle and medial tibeal plateau.
Treatment: Local autologous composition injections into left knee x3, a week
apart.
Result: Five month follow up visit: patient reports about 80% improvement in
symptoms,
no swelling, VAS is 20.
Case 4: J, 26 years, professional swimmer.
Diagnosis: Status post paraspinal muscle injury, chronic paraspinal
tendonitis. Patient
complains of pain over the paraspinal muscle. Prolonged physiotherapy has
shown no
results. Back ROM is full, neurovascular exam is normal. Area of right
paraspinal
hyperemia is identified on colc Doppler Figure 3a), VAS is 80.
Treatment: Inflamed area was marked using the ultrasound technique, autologous
composition was injected intramuscular x 4, a week apart.
Figure 3 is Doppler ultrasound image that reveals a hyperemia in the right
paraspinal area
before treatment (a) and normal post-treatment condition (b).
Result: Doppler shows no inflammation (Figure3 b), VAS is 10 four months after
the
therapy.
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Case 5: S, 18
Diagnosis: Status post right Ailles tear. The patient complains of pain in the
Achilles
insertion: chronic Achilles tendonitis and tenosynovitis. Doppler shows severe
intra-
tendon hyperemia in the area (Figure4a). VAS is 60.
Treatment: Prolonged Physiotherapy and chiropractic treatment didn't reveal
any positive
result. Autologous composition was injected into tendon x3, a week apart under
the
ultrasound guidance.
Figure 4 is a Doppler ultrasound image showing chronic achilles tendinosis
characterized
by excessive hyperemia (a, pre-treatment status), that was resolved as a
result of
autologous composition treatment (b, post-treatment imaging).
Result: no hyperemia on Doppler imaging (Figure 4b), VAS is 0 on five months
post-
injection follow up assessment.
Example 3 ¨ In-Vitro Studies
The study was approved by the Ins 'tutional Review Board. All patients gave
their written
informed consent. Peripheral blood from healthy male and female volunteer
donors (21 to 60
years old) was collected by venipuncture under sterile conditions to the
sterile 10 ml glass tubes.
One tube was manipulated according to a standard procedure with no incubation
step (control
sample). Samples were exposed to different incubation conditions and 500 ul of
serum from each
sample was used for an assay. Samples were centrifuged at 10,000 x g for 10
min in 4C prior to
analysis to remove cell debris and aggregates. BioPlex Pro Human Cytokine 27-
plex Panel,
Human TIMP Magnetic Luminex Performance Assay 4-plex Panel (Bio-Rad
Laboratories,
Canada, LTD) analysis with MagPlex beads was performed in a flat bottom
microtiter plate
according to the manufacturer's instructions
(http://www.hio-
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rad.com/webroot/web/pdf/lsr/literature/10014905.pdf). Briefly, samples were
diluted 1:4 in
sample diluent. Standard was reconstituted and diluted in a fourfold dilution
series. Antibody
coupled capture beads were prepared and plated. The bead solution was vortexed
before addition
to each well. The plate was washed, wash
steps were performed manually. First, the wash
solution was added to the plate that was subsequently covered with sealing
tape. The plate was
incubated on a shaker for 30 s at 1100 rpm and then for 1.5 min at 300 rpm.
The plate was taken
off the shaker and was incubated on a magnet for 1 min before the supernatant
was discarded.
After washing, diluted samples and standards were added in duplicates to the
beads in the wells.
The plate was incubated on a shaker for 30 minutes and after incubation and
washing, detection
antibodies were added for 30 minutes to each well. The plate was again
incubated on a shaker
and after another washing step, streptavidin solution was added for 10 minutes
to the wells. After
a last incubation step, the beads were re-suspended in assay buffer and the
plate was read with a
MagPix (Luminex Corporation) using the xPONENT software (Luminex Corporation,
Austin,
TX, USA). The results were analyzed using the xPONENT software. The absolute
concentrations of the samples were determined by the construction of a
standard curve for each
analyte.
Statistical analysis: Al! statistical tests were performed using GraphPad
Prism version 5.01.
Statistical comparisons were performed using analyses of variance (ANOVA) with
Bonferroni
post-test tests for comparisons between groups. The number of analyzed
experiments was > 3,
and data were shown as mean s.e.m., p< 0.05
Figure 5 is graph showing a compariµ ,n of the level of TIMP 1, TIMP 2 and
TIMP 4 (MMPS
antagonists) proteins in the human serum samples before (baseline level) and
after incubation in
37C for 24h A two-way Anova test reveals a statistically significant increase
in the levels of
TIMP 1 and TIMP 2 at 24 hours post incubation.
Figure 9 is graph showing a comparison between the average concentration level
of PGDF in the
unprocessed human serum samples and an average level of PGDF in 24h incubated
samples T-
test data analysis shows statistically significant increasing of PGDF protein
concentration in
processed samples. PDGF concentration was evaluated using MAGPIX Luminex
technology.
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Example 4 - Results from Eight Patients Treated for Knee Osteoarthritis Joint
Pain
Eight patients were treated for symptoms of knee osteoarthritis joint pain
commencing on
December 3, 2014 according to the method of the present invention. The
patients were provided
with four injections one week apart.
The patients were assessed after with Visual Analog Pain Scale (VAS) and the
Western Ontario
and McMaster Universities Arthritis Index (WOMAC) questionnaire for assessing
pain, stiffness,
and physical function in patients with hip and / or knee osteoarthritis . The
analysis of the
WOMAC questionnaire data showed a statistically significant improvement in the
patient's daily
activities and not significant but positive dynamic of improvement in pain and
stiffness
parameters for the prolonged period (up to 3 months) as shown in figure 7). A
statistical analysis
of Visual Analog Pain Scale revealed significant pain reduction after 3rd
injection as shown in
figure 6.
Figure 6 is a bar graph showing comparison of an average baseline and post
injections of joint
pain according to the VAS scale in the eight patients according to the method
of the present
invention. The results show a statistically significant average decrease in
pain with stable effect
up to 3 months.
Figure 7 is a plot showing point values according to the WOMAC index for
average levels of
pain, stiffness and daily activity capabilities among the eight patients
tested. Values are provided
for a baseline, after injections, two months and three months post injections.
Figure 8 shows results in the form of a graph among the nine patients tested
providing a
breakdown of the average results after the first, second, third injections,
two months and three
months post the fourth injection.
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Although the invention has been described with reference to illustrative
embodiments, it is to be
understood that the invention is not limited to these precise embodiments.
Numerous
modifications, variations, and adaptations may be made to the particular
embodiments of the
invention described above without departing from the scope of the invention.
The scope of the
claims should not be limited by the proferred embodiments set forth in the
examples, but should
be given the broadest interpretation consistent with the description as a
whole.
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References
1. Fernandes JC, Martel-Pelletier J, Pelletier JP. The role of cytokines in
osteoarthritis
pathophysiology. Biorheology. 2002;39(1-2):237-46.
2. Fredberg U, Stengaard-Pedersen K. Chronic tendinopathy tissue pathology,
pain
mechanisms, and etiology with a special focus on inflammation. Scand J Med Sci
Sports.
2008 Feb;18(1):3-15.
3. Loell I, Lundberg IE. Can muscle regeneration fail in chronic inflammation:
a weakness
in inflammatory myopathies? J Intern Med. 2011 Mar;269(3):243-57.
4. Rita E, Mirza, Milie M. Fang,1 William J, Timothy J. Koh. Blocking
Interleukin-113
Induces a Healing-Associated Wound Macrophage Phenotype and Improves Healing
in
Type 2 Diabetes. Diabetes. 2013 Jul; 62(7): 2579-2587.
5. Eming SA, Krieg T, Davidson JM. Inflammation in wound repair: molecular and
cellular
mechanisms. J Invest Dermatol. 2007;127:514.
6. Dinarello CA. Interleukin-1, interleukin-1 receptors and interleukin-1
receptor antagonist.
Int Rev Immunol. 1998;16(5-6):457-99.
7. Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman RL. Two types of
murine helper T cell clone. I. Definition according to profiles of lymphokine
activities
and secreted proteins. J Immunol. 1986 Apr 1;136(7):2348-57.
8. Brandtzaeg P1, Osnes L, Ovstebo R, Joo GB, Westvik AB, Kierulf P. Net
inflammatory
capacity of human septic shock plasma evaluated by a monocyte-based target
cell assay:
identification of interleukin-10 as a major functional deactivator of human
monocytes. J
Exp Med. 1996 Jul 1;184(451-60.
9. Clarke C.11, Hales A, Hunt A, Foxwell BM. IL-10-mediated suppression of TNF-
alpha
production is independent of its ability to inhibit NF kappa B activity. Eur J
Immunol.
1998 May;28(5):1719-26.
10. de Waal Malefyt R, Figdor CG, Huijbens R, Mohan-Peterson S, Bennett B,
Culpepper J,
Dang W, Zurawski G, de Vries JE. Effects of IL-13 on phenotype, cytokine
production,
and cytotoxic function of human monocytes. Comparison with IL-4 and modulation
by
IFN-gamma or IL-10. J Immunol. 1993 Dec 1;151(146370-81.
11. Alvarez RH1, Kantarjian HM, Cortes JE. Biology of platelet-derived growth
factor and
its involvement in disease. Mayo Clin Proc. 2006 Sep;81(9):1241-57.
12. Roberts AB, Flanders KC, Kondaiah P, Thompson NL, Van Obberghen-Schilling
E,
Wakefield L, Rossi P, De Crom-Brugghe B, Heine UI, Sporn MB: Transforming
growth
factor beta: biochemistry and roles in embryogenesis, tissue repair and
remodeling, and
earcinogenesis. Rec Prog Horm Res 44:157-197,1988
13. Roberts AB, Heine UI, Flanders KC, Sporn MB: TGF-beta: Major role in
regulation of
extracellular matrix. Ann NY Acad Sci. "Structure, Molecular Biology and
Pathology of
Collagen". 580:225-232,1990.
-16-
CA 02891445 2015-05-14
14. Gospodarowicz, D., Abraham, J. A., and Schilling, J. Isolation and
characterization of a
vascular endothelial cell mitogen produced by pituitary-derived folliculo
stellate cells.
Proc. Natl. Acad. Sci. USA 86, 7311-7315, 1989.
15. Hirata H, Nagakura T, Tsujii M, Morita A, Fujisawa K, Uchida A.The
relationship of
VEGF and PGE2 expression to extracellular matrix remodelling of the
tenosynovium in
the carpal tunnel syndrome.J Pathol. 2004 Dec;204(5):605-12.
16. Peng H, Usas A, Olshanski A, Ho AM, Gearhart B, Cooper GM, Huard J.VEGF
improves, whereas sFlt1 inhibits, BMP2-induced bone formation and bone healing
through modulation of angiogenesis.J Bone Miner Res. 2005 Nov;20(11):2017-27.
17. Poutsiaka DD, Clark BD, Vannier E, Dinarello CA. Production of interleukin-
1 receptor
antagonist and interleukin-1 beta by peripheral blood mononuclear cells is
differentially
regulated. Blood. 1991 Sep 1;78(5):1275-81.
18. Fufa D, Shealy B, Jacobson M, et al. Activation of platelet-rich plasma
using soluble
Type I collagen. J Oral Maxillofac Surg 2008;66:684-90.
-17-
